It is the broad, long-term goal of this project to understand, on a molecular level, how the p210- bcr-abl protein tyrosine kinase activity ultimately causes a myeloid expansion in chronic phase Chronic Myelogenous Leukemia (CML), and to search for selective therapies efficacious for the treatment of CML. In order to accomplish this, the following Aims are proposed: (1) Expression of Bcr-Abl and its substrates in human primary hematopoietic cells will be determined during hematopoietic differentiation and maturation. CD34+ normal and CML progenitor cells and subpopulations will be stimulated with single and multiple cytokines that induce proliferation and maturation. Expression of Bcr-Abl and its substrates will be assessed and effects of specific inhibitors. (2) The role of SHIP-1 and SHIP-2 in Ber-Abl signaling will be examined. SHIP proteins will be constitutively targeted to the plasma membrane and the effects on Bcr-Abl mediated signaling will be determined in Rat1p210 cells. (3) The role of Dok-1 in c-kit signaling will be determined. The abilities of wt and mutant forms of Dok proteins to interfere with c-kit signaling will be analyzed. The kinase(s) that phosphorylates Dok during c-kit stimulation will be identified as well as the site(s) of tyrosine phosphorylation. (4) Binding partners for SHIP proteins in normal and Bcr-Abl expressing hematopoietic cells will be isolated and identified. (5) Analogues of PD173955, a potent inhibitor of Bcr-Abl kinase, will be analyzed in vitro and in vivo. In collaboration with Drs. William Bornmann and John Kuriyan, we will design and synthesize PD173955 analogues and test their activity in hematopoietic cells in vitro and in animal models of CML.